The multi-PI proposal is to investigate how LRP4 regulates the formation of the neuromuscular junction (NMJ), a synapse that has contributed greatly to understanding of synaptogenesis and of neuromuscular disorders. NMJ formation requires precise interaction between motoneurons (MNs) and muscle fibers. In advance of MN arrival, muscle fibers are prepatterned with small, primitive AChR clusters, which are believed to be critical for NMJ formation although genetic evidence is lacking at the moment. In an established pathway, MNs release agrin to promote postsynaptic development. It binds to LRP4 which acts in cis to stimulate the receptor tyrosine kinase MuSK; and ensuing signaling events lead to AChR clustering. However, little is known of how signals are transduced from agrin to MuSK. In preliminary studies, we solved the first crystal structure of an agrin-LRP4 complex, which provides insight into the initial step of the agrin signaling cascade. Our studies of cell-specific knockout (KO) and double KO mice revealed novel functions of LRP4 in NMJ formation. For example, muscle LRP4 may be critical for presynaptic differentiation during initial steps of NMJ formation. On the other hand, MN LRP4 may serve as agrin's receptor in trans to induce AChR clusters. Many of these findings are unexpected and raise critical questions. How does LRP4 relay the signal from agrin to MuSK? Is MN LRP4 sufficient to induce NMJ formation? Is muscle fiber prepatterning critical for NMJ formation? Is the synaptogenic activity critical for NMJ formation? How does muscle LRP4 regulate motor nerve terminal differentiation? To address these questions, we will 1) understand how signal is transduced from LRP4 to MuSK by solving the structure of the agrin- LRP4-MuSK complex; 2) determine if MN LRP4 is sufficient to induce NMJ formation and if muscle fiber prepatterning is necessary for NMJ formation; and 3) investigate mechanisms by which LRP4 in muscle cells controls presynaptic differentiation. The research represents a synergistic strategy that leverages complementary expertise, strengths and existing resources of the two labs. Results will provide a better understanding of cellular as well as molecular mechanisms of mammalian NMJ formation. Pathogenesis of neuromuscular disorders is known to involve abnormal NMJ structure and function. In fact, mutations of agrin/LRP4/MuSK signaling proteins have been implicated in congenital myasthenic syndrome (CMS). Recent evidence from various laboratories including ours indicates that patients with myasthenia gravis (MG) develop antibodies against MuSK and LRP4. Therefore, our research will contribute to a better understanding of pathogenic mechanisms of these neuromuscular disorders.